Association of end‐stage renal disease with HLA phenotypes and panel reactive antibodies in patients awaiting renal transplantation in Hunan Province

Abstract Objectives To explore the immune‐related genetic susceptibility of human leukocyte antigen (HLA) alleles and their correlation with panel reactive antibody (PRA) generation during end‐stage renal disease (ESRD) progression. Materials and methods Data of the expression patterns of HLA‐A, ‐B, and ‐DR alleles and PRAs of 347 ESRD patients awaiting renal transplantation in Hunan Province from 2015 to 2019 were retrospectively studied. The polymerase chain reaction with sequence‐specific primers was used for HLA genotyping and the enzyme‐linked immunosorbent assay for PRA detection. SPSS 21.0 software was used for all allele frequency and statistical analyses. Results Thirteen HLA‐A, 25 HLA‐B, and 13 HLA‐DR alleles were expressed. The allele frequencies of HLA‐A2, ‐B48, ‐B52, and ‐B55 were significantly higher in the case group than in the control group (p < 0.05), whereas that of HLA‐B60 was significantly higher in the control group (p < 0.05). The frequency of HLA alleles in the PRA‐positive group was significantly higher in females than in males (p < 0.05). The allele frequencies of HLA‐A2, ‐B38, and ‐B46 were significantly higher in the PRA‐positive group than in the PRA‐negative one (p < 0.05), whereas that of HLA‐60 was significantly higher in the PRA‐negative group (p < 0.05). Conclusion HLA‐A2, ‐B48, ‐B52, and ‐B55 may be the ESRD susceptibility alleles in Han Chinese patients in Hunan Province, whereas HLA‐B60 may be the protective allele. Patients carrying HLA‐A2, ‐B38, and ‐B46 are more likely to develop PRA positivity, whereas the opposite is true for those with HLA‐B60.

related to the immune response. 2 Panel reactive antibodies (PRAs) are anti-HLA antibodies produced in the serum of patients. If the serum PRA of a kidney transplant recipient is greater than 10%, then that person is considered to be PRA positive, meaning that the patient is sensitized and the risk of kidney transplantation failure will be high. 3 Because of the nonrandom expression patterns and linkage imbalance of HLA alleles, many studies have determined that the HLA phenotype of any individual has a certain correlation with that person's habitat region, race, disease occurrence, and PRA generation. 4,56 It has also been shown that the long-term survival rate of a kidney graft increases if the HLA types match to a higher degree. 7 To determine the immune-related genetic susceptibility of HLA alleles and their correlation with PRA generation during ESRD progression, in this study, we performed a retrospective statistical analysis using HLA phenotype and PRA data of 347 Han Chinese patients awaiting kidney transplantation in Hunan Province from 2015 to 2019. The results of this study will help toward ensuring that the generation of PRAs and the occurrence of transplant rejection are prevented for patients carrying susceptibility genes for ESRD, thereby improving the success rate of kidney transplantation.

| Patients
Patients with ESRD who were admitted to Xiangya Hospital of Central South University from 2015 to 2019 to await kidney transplantation in Hunan Province were selected for the study.
The exclusion criteria were patients undergoing transfusion, those who had received transplants, and those with incomplete medical information. In total, the data of 347 patients were retrospectively analyzed as the case group. This group comprised 225 males and 122 females in the age range of 14-70 years (average age: 40.6 ± 11.3 years). On the basis of the PRA results, these 347 patients were further divided into a PRA-negative group (n = 310 patients) and a PRA-positive group (n = 37 patients). Additionally, 309 healthy individuals were randomly selected as the control group to determine the HLA allele distribution in a healthy popula-

| Reagents and instruments
The QIAamp DNA Blood Mini Kit was from QIAGEN Biotechnology Co., LTD.; the Micro SSP TM HLA Typing Kit and Lambda Tray TM HLA-specific IgG Antibody Detection Kit were from One Lambda Biotechnology Co., LTD.; and the polymerase chain reaction (PCR) system was from Biometra Co., LTD.

| DNA extraction
Approximately 3 ml of blood was collected intravenously from each participant into a vial containing EDTA as the anticoagulant. The QIAamp DNA Blood Mini Kit was then used to extract DNA from the peripheral blood lymphocytes. The DNA purity was determined to ensure that the A 260 nm /A 280 nm was 1.6-1.8, and the DNA concentration was measured to be 40-100 ng/μl.

| PCR amplification
The Biometra T1 Thermocycler was used for PCR amplification of the isolated DNA. The amplification cycles were set as follows: an initial 130 s at 97°C and 60 s at 63°C; then nine cycles of 10 s at 94°C and 60 s at 63°C; and finally 20 cycles of 10 s at 94°C, 50 s at 59°C, and 30 s at 72°C.

| Electrophoresis of the PCR amplification products
The amplification products were separated on a 2% gel by electrophoresis at 150 V and 50 mA for 5 min. Thereafter, the bands were imaged using a UV analyzer, and One Lambda analysis software was used to determine the genotype.

| ELISA screening for specific IgG antibody
The enzyme-linked immunosorbent assay (ELISA) was performed with the HLA-specific IgG Antibody Detection Kit according to the instructions of the manufacturer. The data were read using an ELISA tester, and the results were analyzed with One Lambda analysis software.

| Statistical analysis
The HLA-A, -B, and -DR allele frequencies were calculated directly.
The Chi-squared test was used to calculate the χ 2 and p values, with a p value of less than 0.05 considered statistically significant.
Relative risk (RR) values and odds ratios were calculated according to the Woolf formula. If the RR value is greater than 1, the allele is considered to be a risk factor for ESRD. If the RR value is less than 1, the allele is considered to play a protective role against ESRD development. If the RR value is equal to 1, the allele is not associated with ESRD. All statistical calculations were performed using SPSS 21.0 statistical software.

| Comparison of HLA allele frequencies between the case and control groups
The HLA-A, -B, and -DR genotypes were analyzed in 347 patients whose primary ailment was kidney disease that eventually progressed to ESRD.

| Allele frequency of the HLA-A locus
In total, 13 HLA-A alleles were detected in the case group, which had more HLA-A29 and HLA-A74 alleles (frequency: 0.14% and 0.29%, respectively) than the control group. By contrast, the control group had a higher frequency of the HLA-A23 allele (0.32%). In both groups, the first three alleles were HLA-A11, -A2, and -A24, which occurred at a frequency of 37.03%, 32.28%, and 17.58%, respectively, in the case group, and 39.32%, 36.21%, and 18.12%, respectively, in the control group. The frequency of HLA-A2 in the case group was significantly higher than that in the control group (p < 0.05), whereas there was no statistically significant difference in the frequencies of the other alleles between the two groups (p > 0.05). The allele frequency results for this locus are presented in Table 1.

| Allele frequency of the HLA-B locus
In total, 25 HLA-B alleles were detected in the case group, which had a higher frequency of HLA-B76 (0.43%) than the control group.
In both groups, the first three alleles detected were HLA-B60, -B46, and -B13, occurring at the frequency of 20.17%, 17.72%, and 8.50%, respectively, in the case group, and 25.57%, 16.99%, and 10.68%, respectively, in the control group. The frequencies of the HLA-B48, -B52, and -B55 alleles were significantly higher in the case group (p < 0.05), whereas that of the HLA-B60 allele was significantly lower (p < 0.05). There were no statistically significant differences in the frequencies of the other alleles between the two groups (p > 0.05).
The allele frequency results for this locus are presented in Table 2.

| Allele frequency of the HLA-DR locus
In total, 13 HLA-DR alleles were detected in the case group, in which the top three alleles HLA-DR09, -DR04, and -DR12 were detected at a frequency of 17.29%, 15.56%, and 13.40%, respectively. Among the HLA-DR alleles detected in the control group, the top three were HLA-DR09, -DR15, and -DR04, occurring at a frequency of 20.87%, 13.27%, and 12.46%, respectively. However, there were no statistically significant differences in allele frequencies found between the two groups for the HLA-DR locus. The allele frequency results for this locus are presented in Table 3.    Table 4.

| DISCUSS ION
As a very important genetic marker of human beings, the HLA system comprises a group of genes that determine whether the transplanted tissue is compatible with the recipient's HLA phenotype and is closely related to the immune response. 8 For patients who progress from primary nephropathy to ESRD and require kidney transplantation, the degree of match or mismatch of the HLA types will directly affect the success rate of organ transplantation and subsequent immunosuppressive therapy. 9 According to "China's Common and Welldocumented Alleles, and Confirmed HLA Allele  Many studies have shown that the immune-related genetic susceptibility of the HLA system is associated with various diseases, and many HLA genes have also been reported to be associated with the RR of primary kidney disease. 12 In the PRA-positive group of this study, the HLA allele frequency in the females was 22.95%, which was significantly higher than that in the males (4%; p < 0.05), indicating that there were statistically significant differences in PRA production between the different gender groups. The reason that the female patients were more likely to develop PRAs may be due to an increased exposure to foreign antigen stimulation during pregnancy, and this difference is consistent with that described in other studies. 18 and -B46 were the first two alleles detected in the HLA-B locus in this study, their frequencies differed in the PRA-positive and -negative groups. This allele frequency difference was also found in another local study. 20 The association of HLA-B60 with a significantly reduced risk of sensitization has been reported. 21 The inference about the correlation between HLA alleles and PRA generation in this study-that is, patients with HLA-A2, -B38,

| CON CLUS ION
In

ACK N OWLED G EM ENTS
The authors have not received any funding or grants in support of the presented research or for the preparation of this report and have no potential conflicts of interest to declare. This study was conducted under the criteria set forth in the Declaration of Helsinki and was approved by the Clinical Medical Ethics Committee, Xiangya Hospital, Central South University.

AUTH O R CO NTR I B UTI O N S
QS contributed to study conceptualization, sample processing, interpretation of results, and writing-first draft of manuscript and final manuscript approval. LL: statistical analysis, study design, and interpretation of results. Both authors have reviewed and approved the final version of the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing was not applicable to this article, as no datasets were generated or analyzed during the current study.